The long term goal of the proposed research is to elucidate the anatomical, physiological and neurochemical mechanisms that control LC activity. Using a multidisciplinary approach, the proposed studies address specific hypotheses generated from the results of the current period of support, and aim to resolve significant issues concerning LC function. Specifically, our previous results have generated important questions concerning (i) inputs to LC-projecting neurons in the rostral medulla, (ii) the role of neurons in the pericoerulear area in the control of LC activity, (iii) the origin and regulatory influences of neuropeptides that innervate LC neurons, and (iv) inputs to the nucleus LC vs. to extranuclear LC dendrites. These are the next most significant issues for understanding afferent control of LC and, more generally, for arriving at a better understanding of LC function. Our recent results indicate that LC-projecting neurons in PGi and PrH are widely but topographically disturbed, and that afferents to PGi are located in several brainstem areas, with considerable topography present for their terminal fields with PGi. However, we do not know from this analysis which circuits projecting to PGi or PrH are influencing LC-projecting neurons. Using light as well as electron microscopic analysis, we will identify these second order afferents involved in LC control. Companion neurophysiologic studies will confirm these anatomic results, and determine the functional impact of these second-order afferent projections on LC activity. Many areas that were thought to innervate LC instead innervate specific regions in the pericoerulear area. We have recently analyzed the pericoerulear region, finding that it is a cell-rich region with a variety of neurotransmitters located in constituent neurons. However, it is not known whether peri-LC neurons project to LC neurons. This possibility would provide a local circuit whereby areas that project to the pericoerulear region may indirectly influence LC cells. We will determine the anatomic relationship between pericoerulear regions and noradrenergic LC neurons. Several lines of evidence indicate that neuropeptides play an important role in LC regulation. However, the source neurons for these peptide projections to LC are unknown, and there has been no systematic investigation of possible physiologic interactions of neuropeptides with the major inputs to LC from Pgi and PrH. Our recent finding that these two nuclei provide excitatory amino acid and GABAergic inputs to LC now open the way for detailed analysis of the regulatory role of peptides on LC activity. We will identify the sources of these neuropeptides in LC afferent areas, and determine the effects of neuropeptides on spontaneous and PGi, PrH, and sensory-evoked activity in LC. Our recent analysis reveals that LC dendrites extend beyond the nucleus in specific rostromedial and caudal zones. These zones receive inputs from specific regions that do not innervate the LC proper. However, its is not known if these projections contact LC dendrites or non-LC neurons. We will determine if such inputs contact LC dendrites or pericoerulear neurons. We will also characterize the innervation of LC neurons by pericoerulear and peptide inputs identified above, to determine if they contact the somata or extranuclear dendrites of LC neurons. These studies are the next steps needed in our comprehensive analysis of circuits that control LC, extending our investigation to the ultrastructural and multisynaptic levels. They will also provide the first systemic investigation of neuropeptide innervation and function in LC. A better understanding of the afferent control of LC will provide important insights and constraints for future ideas of the functional significance of this preeminent brain noradrenergic nucleus.